日本血吸虫Tsunagi样蛋白编码基因功能的鉴定

发布时间：2018-11-18 13:31

【摘要】： 血吸虫病是严重危害人民身体健康,阻碍社会经济发展的重大传染病。日本血吸虫致病主要是由于虫卵在肝脏大量沉积形成的虫卵肉芽肿及其纤维化。控制血吸虫性别分化、性成熟及雌虫产卵成为防治血吸虫病的重要策略之一。为了解日本血吸虫Tsunagi蛋白的功能,我们用RNAi技术下调Tsunagi基因的转录水平,再观察干扰作用后虫体的发育(尤其是生殖系统发育)是否与正常虫体之间存在差异。按照双链RNA(dsRNA)体外合成试剂盒要求,我们从日本血吸虫童虫cDNA文库中扩增出Tsunagi基因,将其片段正、反义链连接上T7启动子,然后把连接有T7启动子的目的基因的正、反义链进行PCR扩增。将PCR产物转录成单链RNA(ssRNA)并将其纯化,最后把一对ssRNA混合合成dsRNA。阴性对照组我们采用试剂盒中提供的基因。在BTX电穿孔仪上,设定参数为125V电压、20ms脉冲时值、1次脉冲次数,将dsRNA电穿孔转染入机械制备的日本血吸虫童虫体内,体外培养于电转后第1, 3, 5天收集童虫,按TRIzol方法同时提取童虫的总RNA及总蛋白。经实时荧光定量PCR及Western blotting分别检测Tsunagi基因和蛋白表达水平变化。结果发现Tsunagi基因的转录水平实验组分别于电穿孔后第1, 3, 5天较阴性对照组下降16%,57%,75%。该基因的蛋白水平在第1, 3, 5天较阴性对照组下降15%,38%,52%。实验结果证明dsRNA可以特异性的抑制日本血吸虫靶基因以及蛋白的表达且效果明显。经dsRNA电转的童虫注射入小鼠体内,6周后取出虫体通过一系列处理制成标本,在激光共聚焦显微镜下观察虫体内部各器官形态特征及测量虫体体长、体宽、睾丸长、睾丸宽、睾丸面积、卵巢长、卵巢宽、卵巢面积。结果发现SjTsunagi dsRNA组中8条中3～4条雄虫睾丸内有大量精子出现,而雌虫中卵巢,卵黄腺中却没有明显特征变化;测得各项指标经SPSS 13.0统计软件分析阴性对照组与SjTsunagi dsRNA组的体宽、睾丸长、睾丸宽、睾丸面积、卵巢长、卵巢宽、卵巢面积均有显著性差异。由此结果我们得出Tsunagi基因在日本血吸虫中是生殖相关基因,在生殖系统器官的正常发育起一定作用。
[Abstract]:Schistosomiasis is a major infectious disease that harms people's health and hinders social and economic development. Schistosoma japonicum is mainly caused by egg granuloma and fibrosis. Controlling the sex differentiation, sexual maturation and laying eggs of female schistosomiasis is one of the important strategies to control schistosomiasis. In order to understand the function of Tsunagi protein in Schistosoma japonicum, we down-regulated the transcription level of Tsunagi gene by RNAi technique, and then observed whether there were differences between the body development (especially reproductive system development) and normal body after interference. According to the requirement of double-stranded RNA (dsRNA) in vitro synthesis kit, we amplified the Tsunagi gene from the cDNA library of Schistosoma japonicum, and ligated the Tsunagi gene into T7 promoter by sense, antisense chain, and then ligated the target gene with T7 promoter. Antisense strand was amplified by PCR. PCR products were transcribed into single-stranded RNA (ssRNA) and purified. Finally, a pair of ssRNA was mixed to synthesize dsRNA.. In the negative control group, we used the genes provided in the kit. On the BTX electroporation instrument, the parameters of 125V voltage, 20ms pulse time value and the number of pulses were set. The dsRNA electroporation was transfected into the mechanically prepared Schistosoma japonicum child worm and cultured in vitro on the 1st, 3rd and 5th day after electroporation. Total RNA and total protein were extracted by TRIzol method. The expression levels of Tsunagi gene and protein were detected by real-time fluorescence quantitative PCR and Western blotting, respectively. The results showed that the transcription level of Tsunagi gene in the experimental group was 16% lower than that in the negative control group on the 1st, 3rd and 5th day after electroporation. The protein level of the gene was 15% lower than that of the negative control group on the 1st, 3rd and 5th day compared with the negative control group. The results showed that dsRNA could specifically inhibit the expression of target gene and protein of Schistosoma japonicum. The mice were injected with dsRNA electroporated baby worms. After 6 weeks, the worm bodies were taken out and made into specimens by a series of treatments. The morphological characteristics of organs in the body were observed under confocal laser microscope, and the length, width, length of testis and width of testis were measured under confocal laser microscope. Testicular area, ovarian length, ovarian width, ovarian area. The results showed that a large number of spermatozoa were found in the testis of 3 male and 4 males in the SjTsunagi dsRNA group, but there were no obvious changes in the ovaries and yolk glands in the females. The body width, testicular length, testicular width, testicular area, ovary length, ovarian width and ovarian area were significantly different between the negative control group and the SjTsunagi dsRNA group by SPSS 13.0 statistical software. It is concluded that Tsunagi gene is a reproductive related gene in Schistosoma japonicum and plays a certain role in the normal development of reproductive system organs.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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